Glutathione (GSH), a tripeptide that is normally found in all animal cells and most plants and bacteria at relatively high (1-10 millimolar) concentrations, helps to protect cells against oxidative damage that would otherwise be caused by free radicals and reactive oxidative intermediates (ROIs) produced during cell metabolism or as the results of, for example, drug overdose. Glutathione is itself the major scavenger of reactive oxidative intermediates present in all eukaryotic forms of life and is generally required to protect cells against damage by oxidants. Glutathione reduces (and thereby detoxifies) intracellular oxidants and is consumed by this reaction. Glutathione is oxidized to the disulfide linked dimer (GSSG), which is actively pumped out of cells and becomes largely unavailable for reconversion to reduced glutathione. Thus, unless glutathione is resynthesized through other pathways, utilization of this compound is associated with a reduction in the amount of glutathione available. The antioxidant effects of glutathione are also mediated less directly by the role of this compound in maintaining other antioxidants in reduced form. Thus, pharmaceutical compounds that replenish or elevate glutathione levels work, at least in part, through enhancement of the defense mechanisms seemingly utilized to normally protect tissue from ROI mediated damage.
Glutathione depletion has been implicated in the pathology of a number of diseases including infection by human immunodeficiency virus (HIV). In HIV infection, cysteine/glutathione depletion is known to impair T-cell function and is associated with impaired survival of subjects with less than 200 CD4 T-cells per μl blood.
Drug toxicity is a very widespread problem. Cysteine/glutathione depletion and oxidative stress (See U.S. Pat. No. 4,757,063) intensify drug toxicity effects and have been implicated in the mechanism of drug toxicity reactions.
For example, acetaminophen is known to act to deplete cysteine/glutathione and cause a variety of drug toxicity symptoms. Acetaminophen, also known as paracetamol and N-acetyl-p-aminophenol, is one of the most widely used pharmaceutical analgesic and antipyretic agents in the world. It is contained in over 100 products and is commonly found in the U.S. as immediate release tablets and as extended-release preparations. Various children's chewable, suspension, and elixir formulations that contain acetaminophen are prevalent. Acetaminophen is also found as a component of combination drugs, such as propoxyphene/acetaminophen and oxycodone/acetaminophen.
Acetaminophen continues to be the most commonly encountered substance in toxic ingestions. In many cases, acetaminophen overdoses are unintentional and are undiagnosed until after substantial damage has already occurred. Repeated administration of acceptable size doses of acetaminophen can produce toxicity symptoms. As discussed by Donovan (1999) Academic Emergency Med. 6:1079-1082, methods for detecting post-ingestion blood levels of acetaminophen suffer from poor predictive values. Even in the simple case of a single acute ingestion, patients with no discernible risk factors for liver injury and low blood levels of acetaminophen still develop toxicity and even die.
Many companies package acetaminophen under different trade names, resulting in inadvertent overdosing by less sophisticated patients and parents who do not read the information on the packaging. In addition, cold remedies and other over-the-counter preparations often contain acetaminophen, which is listed among a series of generic drug names that are difficult for patients and parents to read. Therefore, patients often are unaware of the amount of acetaminophen that they have received. Children are especially vulnerable to accidental exposure due to their smaller size, the presence of acetaminophen in multiple over-the-counter remedies, and a reluctance to administer aspirin and other NSAIDs to children for fever due to the risk of Reye's Syndrome and renal tubular injury. The antipyretic value of acetaminophen clearly has been demonstrated and hence acetaminophen is widely used in hospitals for this purpose. However, acetaminophen may not be the antipyretic agent of choice under circumstances where renal or hepatic function is in danger of being compromised.
It is well established that large acetaminophen overdose causes hepatotoxicity and, in some cases, nephrotoxicity in humans and in experimental animals. Acute overdosage of acetaminophen results in dose-dependent and potentially fatal hepatic necrosis as well as (in rare cases) renal tubular necrosis and hypoglycemia. Acetaminophen is rapidly absorbed from the stomach and small intestine and is normally metabolized by conjugation in the liver to nontoxic agents, which are then eliminated in the urine. In acute overdoses, or when maximum daily doses are exceeded over a prolonged period, the normal pathways of metabolism become saturated.
Excess acetaminophen is metabolized in the liver via the mixed function oxidase P450 system to a toxic, N-acetyl-p-benzoquinone-Imine (NAPQI). NAPQI has an extremely short half-life and is rapidly conjugated with glutathione, a sulfhydryl donor, and removed from the system. Under conditions of excessive NAPQI formation or reduced glutathione stores, NAPQI is free to bind to vital proteins and the lipid bilayer of hepatocytes. This results in hepatocellular death and subsequent centrilobular liver necrosis. Immunohistochemical studies have suggested that NAPQI-protein adducts appear even at sub-hepatotoxic acetaminophen doses and before depletion of total hepatic glutathione which may be related to rare cases of hypersensitivity. In addition, decreased intracellular cysteine/glutathione can contribute to cell death via mechanisms that do not involve NAPQI.
The direct cost of acetaminophen overdose has been estimated to be $87 million annually. Effective protocols have been developed and tested to stratify risk and treat patients who present soon after a single large dose of acetaminophen. However, many patients present after a delay long enough to metabolize all the acetaminophen, after two or more ingestions over several hours, or after several days of excessive self-medication. Under these circumstances it is difficult for the clinician to estimate the risk of adverse outcome before hepatic or renal injury occurs. See, for example, Bond and Hite (1999) Acad. Emerg. Med. 6:1115-1120; and Donovan (1999) Acad. Emerg. Med. 6:1079-1082. However, early treatment of acetaminophen overdosage is considered to be crucial, and vigorous supportive therapy is essential when intoxication is severe.
Nucleoside reverse transcriptase inhibitors (NRTIs), of which the pyrimidine nucleoside analogue azidothymidine (AZT, zidovudine), is a common example, are often given in combination therapies with other anti-retroviral drugs to treat HIV. Long-term therapy with AZT is commonly associated with dose-dependent hematologic toxicity which manifests as low erythrocyte counts and elevated mean red cell volume, and with muscle fiber toxicity, particularly in patients with advanced HIV disease. Some studies indicate that AZT's toxic interactions result from the generation of reactive oxygen species (ROIs) that react with and deplete intracellular glutathione levels. See de la Asuncion, et al (1998) J. Clin. Invest. 102(1): 4-9; Gogu et al. (1991) Exp. Hematol. 19(7): 649-652; Gogu and Agrawal (1996) Life Sci. 59 (16): 1323-1329; Prakash et al (1997) Arch. Biochem. Biophys. 343 (2): 173-80.
Results have shown that acetaminophen usage, which lowers glutathione levels exacerbates AZT toxicity. Richman, et al. (1987) N. Eng. J. Med. 317: 192-97. De Rosa et al. recently have shown that treatment with NAC which increases glutathione levels decreases the toxicity. De Rosa et al., submitted to JAMA for publication.
Evidence from in vitro and animal studies supports this conclusion. AZT treatment caused oxidative damage to mitochondrial DNA (including increased mitochondrial lipoperoxidation) and increased levels of oxidized glutathione in skeletal muscle in mice. See de la Asuncion, et al. (1998) J. Clin. Invest. 102(1): 4-9. NAC and the anti-oxidant Vitamins C and E have been shown to prevent this AZT-induced toxicity. See id.; Gogu, et al (1991) Exp. Hematol. 19, 649-52; and Gogu and Agrawal (1996) Life Chem. Rep. 4, 1-35. Furthermore, AZT treatment intensifies glutathione depletion in HIV-TAT transgenic mice (see Prakash, O., et al. Arch. Biochem. Biophys. (1997) 343: 173-80) where expression of the TAT protein has been shown to deplete glutathione by decreasing glutathione biosynthesis (see Choi, J., et al., (2000) J. Biol. Chem. 275 (5): 3693-98) and the activity of antioxidant enzymes (Flores et al. (1993) Proc. Nat. Acad. Sci. 90 (16): 7632-36). Studies with TAT-transgenic mice also show that AZT toxicity is enhanced in this glutathione-depleted environment. See Prakash et al. (1997) Arch. Biochem. Biophys. 343, 173-80.
Early clinical trials of AZT efficacy in HIV disease revealed an association between AZT toxicity and the use of acetaminophen. See, e.g., Richman, et al. (1987) New Eng. J. Med. 317(4): 192-97. Although the mechanism of this toxic reaction is not fully understood, acetaminophen does not impair AZT detoxification since no difference in AZT's rate of destruction has been observed. Since acetaminophen is known to deplete glutathione, the potentially harmful effect of co-administering acetaminophen and AZT appears to be mediated through glutathione depletion. Thus, in conditions where glutathione already is depleted, such as in later stages of HIV disease, detoxification of acetaminophen (which can be expected to further deplete glutathione stores in the liver and elsewhere) would increase the potential for AZT toxicity.
Long-term antibiotic usage also often produces drug toxicity reactions. Toxicity reactions for a given antibiotic are a function of its mechanism of action and the pathway(s) by which it is metabolically degraded.
Scientists have long sought to identify agents that will be generally effective in combating drug toxicity reactions. Protective agents for drug overdose have been extensively studied. A known method of treatment for acetaminophen overdose is the administration of sulfhydryl compounds. L-methionine, L-cysteine, and either the purified L-enantiomer or the racemate mixture of N-acetylcysteine are known to have a protective action in animals. Methionine and another sulfhydryl compound, cysteamine, have been reported to provide some protection. Also, cimetidine, dimethyl sulfoxide, and ethanol have been shown to inhibit acetaminophen bioactivation. N-acetylcysteine has been shown to be effective in humans when given orally. Early administration of compounds supplying sulfhydryl groups (0 to 10 hours after acetaminophen ingestion) may prevent or minimize hepatic and renal injury in cases of acetaminophen overdose. NAC is now used by many physicians for treatment of hepatic failure of any etiology, whether known or unknown, and is the accepted antidote for cyclophosphamide poisoning. NAC also is used to prevent toxicity due to radiation therapy contrast material in patients undergoing such treatment. The mechanisms through which NAC prevents or reverses toxicity are mainly thought to involve glutathione replenishment. However, additional mechanisms through which NAC works directly via the cysteine molecule itself are not excluded.
Recently we have shown that treatment with NAC decreases AZT's hematologic toxicity in subjects taking AZT. It appears that NAC provides cysteine needed to redress the excessive sulfur loss that occurs in MV disease and specifically to replenish intracellular glutathione. This in turn helps to restore the reducing power necessary for deoxynucleotide synthesis and to bring the size of the deoxynucleotide pool, and hence the rate of cell division, back into normal range. This decreases the AZT-mediated inhibition of erythroid development, helps to improve the overall metabolism and stability of erythrocytes and their progenitors (for example, by enabling optimal functioning of the glucose-6-phosphate dehydrogenase and other energy-supplying pathways). In addition, it improves the cell's ability to withstand the production of oxidants induced by the introduction of drugs (such as AZT) and the internal production of molecules (such as TNF and HIV-TAT) that trigger intracellular oxidant production.
Improved formulations and methods to prevent drug toxicity reactions during long term therapy are of particular interest in view of the considerable loss of life attributable to, and cost of treatment of, such reactions. The present invention addresses this problem.